WO2014093696A2 - Dérivés d'insuline pour le traitement du diabète - Google Patents

Dérivés d'insuline pour le traitement du diabète Download PDF

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WO2014093696A2
WO2014093696A2 PCT/US2013/074794 US2013074794W WO2014093696A2 WO 2014093696 A2 WO2014093696 A2 WO 2014093696A2 US 2013074794 W US2013074794 W US 2013074794W WO 2014093696 A2 WO2014093696 A2 WO 2014093696A2
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insulin
group
derivatized
acid
amine
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PCT/US2013/074794
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WO2014093696A3 (fr
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Daniel G. Anderson
Hung-Chieh Chou
Matthew J. WEBBER
Benjamin C. TANG
Yair Levi
Yunlong Zhang
Rosemary Lynn KANASTY
Arturo Jose VEGAS
Robert A. LANGER
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Massachusetts Institute Of Technology
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Publication of WO2014093696A3 publication Critical patent/WO2014093696A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/62—Insulins

Definitions

  • This invention generally relates to smart or interactive delivery systems for therapeutics, prophylactic or diagnostic agents in response to glucose levels.
  • Diabetes mellitus is a disorder of glucose regulation with accumulation of glucose in the blood.
  • insulin is secreted basally, usually in the range of 0.5 to 1.0 units per hour, and the levels are increased after a meal.
  • the pancreas secretes a bolus of insulin, which returns blood glucose to normal levels by stimulating the uptake of glucose into cells and signaling the liver to reduce glucose production.
  • the early phase (responsible for shutting down hepatic glucose production) is a spike of insulin release that occurs within 2-15 minutes of eating.
  • the late phase release extends about 2 hours. Between meals the liver breaks down glycogen stores to provide glucose to the brain and other tissues.
  • Diabetes results in chronic hyperglycemia due to the inability or reduced ability of the pancreas to produce adequate amounts of insulin or due to the inability or reduced ability of cells to synthesize and/or release insulin.
  • the effectiveness of the first-phase response is decreased or absent, leading to elevated postprandial glucose levels.
  • Diabetes is a major public health problem affecting 285 million people across the world and this number is expected to be over 450 million by 2030 (Wild, et al, Diabetes Care, 27: 1047- 1053 (2004).
  • the malfunction of glucose regulation arises from (1) insufficient secretion of insulin due to autoimmune-mediated destruction of pancreatic ficelle (type 1 diabetes) or (2) disorders of both insulin resistance and secretion (type 2 diabetes) (Pickup, et al, Diabetes Metab. Res. Rev., 24: 604-610 (2008); Stumvoll, et al. Lancet, 365: 1333-1346 (2005); and Kahn, Diabetes 43: 1066- 1084 (1994).
  • pancreas-like, synthetic closed-loop device able to continuously and intelligently release insulin in response to blood glucose levels is highly desirable (Kumareswaran, et al. Expert Rev. Med. Devices, 6:401-410 (2009); Ravaine, et al, J. Control Release, 132:2-11 (2008)).
  • a straightforward strategy to achieve continuous release in response to glucose levels is to integrate a glucose monitoring moiety and a sensor-triggered insulin releasing moiety into one system.
  • a number of glucose- responsive formulations and devices have been explored, mainly derived from three categories: (1) glucose oxidase (GOx) based enzymatic reaction-induced response systems; (2) binding lectin protein Concanavalin A (Con A) based response systems, and (3) phenylboronic acid (PBA) based synthetic glucose- binding systems (Ravaine, et al, J. Control Release 132:2-11 (2008)).
  • Efforts to prepare insulin with patient-specific kinetics have explored a number of different modification strategies to create variants with more rapid activity as well as those with prolonged activity, and combinations of various types may be useful for improved glycemic control.
  • native insulin which forms hexamers when formulated with zinc, has an onset time of 30-60 minutes, a peak window of action from 2-3 hours, and a duration of action of 8-10 hours.
  • Fast acting formulations have been developed, such as Insulin Lispro where the B29 lysine residue and the B28 proline residue have been switched, in order to prevent hexamer formation and improve uptake.
  • Lispro has a reduced onset time of just 5-15 minutes, with its a peak action at 30-90 minutes and a duration of action of 4-6 hours.
  • Long acting formulations such as Insulin Detemir where a saturated fourteen-carbon alkyl segment is covalently attached to the amine side-chain of lysine B29, have been developed to prolong insulin duration by enabling it to bind to and be sequestered by circulating serum albumin.
  • Insulin Detemir has an onset of action at 1-2 hours, with peak action at 6-8 hours and duration lasting up to 24 hours.
  • Long acting insulin in particular, is useful as a daily injection to supplement basal insulin levels and prevent spikes in blood glucose levels throughout the day.
  • PBA is boronic acid containing a phenyl substituent and two hydroxyl groups attached to boron.
  • PBA and its derivatives form complexes with polyol molecules such as glucose and fructose, and can form complexes with polyols and diols.
  • the ability of PBA to bind polyols and diols has been exploited in different ways to provide a glucose binding insulin delivery system. Some researchers have directly coupled a PBA moiety to insulin, to provide glucose binding insulin. For example, U.S. Publication No. 20030186846 by Hoeg-
  • Johnson, et al discloses an insulin delivery system made of insulin derivatives with a built in glucose sensor, such as an aryl boronate moiety.
  • Compounds, compositions, and methods for "smart" delivery of a therapeutic, prophylactic or diagnostic agent, such as glucose-mediated delivery of insulin through glucose-sensing insulin derivatives, are provided.
  • the insulin derivatives bind serum albumin or agglomerate in vivo.
  • the insulin derivatives effectively dissociate to release insulin under hyperglycemic conditions, where the complexation of glucose to a glucose-sensing element alters properties of the insulin derivative leading to the dissociation.
  • the compounds, compositions, and methods provide a delivery strategy for both self-regulated and long-term diabetes management.
  • Other therapeutic, prophylactic, or diagnostic agents can be included or substituted for delivery.
  • derivatives of glucagon, GLP-1, or a GLP-1 agonist can be used.
  • the insulin derivative contains insulin or an insulin analog covalently linked a component containing a glucose binding component.
  • the component containing the glucose binding component includes a hydrophobic group.
  • the derivatized insulin can include one or more phenylboronic acid (PBA) groups, a
  • the hydrophobic group can be covalently linked to the insulin or insulin analog and the PBA groups can be covalently linked to the hydrophobic group, the insulin or insulin analog, or both.
  • the insulin or insulin analog can form insulin hexamers and the derivatized insulin can bind to serum albumin in inverse proportion to glucose levels.
  • the insulin derivative can bind to serum albumin when glucose levels are low or normal and can be released from binding when glucose levels are high.
  • the insulin derivative has the formula Xi— X 2 , where Xi is insulin or an insulin analog and X 2 is a component containing a glucose binding component.
  • the insulin or insulin analog is derivatized with a lipid.
  • the lipid is derivatized with a PBA group.
  • X 2 can be -CO-alkylene-Pv9 or -CO-alkenyl-Rg, where R9 is a
  • the alkylene or alkenyl group can be substituted or unsubstituted.
  • the number of carbons in the alkylene or alkenyl group can vary. In some embodiments, the number of carbons is from about 3 to about 25. In some embodiments, the number of carbons is 11.
  • the insulin or insulin analog is derivatized with a bile acid.
  • the bile acid is derivatized with one or more PBA groups.
  • X 2 can be -CO-R 13 , where R 13 is a bile acid and one or more hydroxyls on the bile acid are derivatized with a phenylboronic acid group.
  • the bile acid is cholic acid, lithocholic acid, hyocholic acid, deoxyxholic acid, hyodeoxycholic acid, or chenodeoxycholic acid.
  • the insulin or insulin analog is derivatized with a pseudolysine-containing group.
  • the pseudolysine-containing group is derivatized with a PBA group.
  • X 2 can be - CO-(CH 2 ) r -NH-CO-CHRi 4 -NH-CO-(CH 2 ) s , where r is an integer from 3-25, s is an integer from 3-25, and R 14 is an amine-containing group comprising a phenylboronic acid group.
  • r is 3, 5, or 11 and s is 6, 8, 10, 12, or 14.
  • r and s together total to an integer from 13 to 21.
  • the insulin or insulin analog is derivatized with a linker with a functional group.
  • the linker is derivatized with a PBA group.
  • the functional group is selected from a variety of functional groups such that agglomeration of the insulin is glucose-responsive.
  • X 2 can be -CO-(CH 2 ) j -NH-CO-CRiR 2 , where j is an integer from 3-25, where Ri is -NH-R 12 or -NH-CO-CH 2 -CH 2 -CNRi 2 -R 32 , where R 32 is glucamine, gluconic acid, glucosamine, fructosamine, galactosamine, mannosamine, or other hexosamines; Ri 2 is selected from the group consisting of hydrogen, -S0 2 alkyl, -S0 2 cycloalkyl, -S0 2 heterocycloalkyl, -S0 2 aryl, -S0 2 heteroaryl, - COalkyl, -COcycloalkyl, -COheterocycloalkyl, -COaryl, -COheteroaryl, - CONHalkyl, -CONHcycloalkyl, -CONHheter
  • the insulin or insulin analog is derivatized with an oligomer of monomer residues having modified side chains. At least one of the side chains is modified with a PBA group.
  • X 2 can be an oligomer of 2 to 5 monomer residues, where the monomers comprise single modified side chains, dual modified side chains, or
  • the side chains are modified with a phenylboronic acid group, hydrophobic residues, hydrophilic residues, charged residues, diol residues, fluorescent residues, and combinations thereof, where at least one of the side chains is modified with phenylboronic acid.
  • each monomer residue of the oligomer is -CO-O- R 3 -, where R 3 is -CR 4 -(CH 2 ) m -NH- or pyrrolidine substituted with R 4 , where m is an integer from 0-25, R 4 is -CO-NH-R 5 or -CO-NH-C(CH-CO-NH-R 5 ) 2 , and R 5 is the side chain modification.
  • R 5 is a phenylboronic acid group, C 8 -i 8 alkyl, -CH 2 -phenyl, -(CH 2 -CH 2 -0) p -H or -(CH 2 -CH 2 -0) p -CH 3 , wherein p is an integer from 1-500, -CH 2 -dioxane, -CH 2 -CH 2 -oxazane, -CH 2 - CH 2 -N(CH 2 -CH 3 ) 2 , -CH 2 -CH 2 -pyrazole, a fluorescent group, -piperidine-phenyl, -piperidine-oxazane, -piperidine-CH 2 -CH 2 -N(CH 2 -CH 3 ) 2 , -piperidine-CH 2 -CH 2 - pyrazole, -dimethylaminobenzyl, or -pyridine. At least one R 5 is a phenyl
  • the insulin or insulin analog is derivatized with a diol-containing group.
  • the diol-containing group is complexed with a PBA group.
  • X 2 can be -CO-R 6 -R 7 , where R 6 is a linker or is not present and R 7 is a diol-containing group complexed to a hydrophobic phenylboronic acid (PBA) group.
  • the diol-containing group includes one or more diols.
  • the hydrophobic PBA group includes one or more PBA groups covalently linked to a hydrophobic group. At least on diol and one hydrophobic PBA group form a boronic ester.
  • the diol-containing group is -(DOPA-Gly)i-NH 2 , wherein i is an integer from 1-5. In some embodiments, the diol-containing group is 6-methyl-6-deoxy-D-galactose, l-deoxy-P-D-lactopyranoside, a-D- Mannopyranosyl, or adenosine. In some embodiments, R 6 is -CO-(CH 2 )h-R3i-, where h is an integer from 3-25 and R 31 is O-triazole- or CO-NH-CH 2 -CO- dibenzo-cyclocta-triazole-. In some embodiments, the hydrophobic group is - (CH2) k -CH 3 , where k is an integer from 3-25. In some embodiments, the hydrophobic group is a bile acid. In some embodiments, k is 11.
  • the glucose binding component is a chemical group capable of binding to or reacting with glucose.
  • Examples of reversible glucose sensors include organic borates, such aryl boronates or other borates.
  • the glucose binding component is a PBA group.
  • the PBA group is:
  • Ri 6 is NH, NR 29 , or is not present; Ri 7 is CH 2 , CO, S0 2 , or is not present; Ri 8 , R 19 , R 2 o, R 21 , and R 22 are each independently -B(OH) 2 , -F, -N0 2 , - CN, -H, or not present, where one and only one of Rig, R19, R20, R21, and R22 is - B(OH) 2 ; R23, R24, R25, R26, and R 27 are each independently C or N, where at most only three of R 23 , R24, R25, R26, and R 27 are N; and R29 is Ci_ 4 alkyl.
  • the derivatized insulin is made by (i) reacting a N- hydroxysuccinimide (NHS)-activated alkyne linker with insulin or an insulin analog to form an alkyne-derivatized insulin, (ii) reacting a diol-containing compound comprising azide with the alkyne-derivatized insulin to form diol- derivatized insulin, and (iii) reacting a hydrophobic phenylboronic acid (PBA) group with the diol-derivatized insulin to form the derivatized insulin.
  • NHS N- hydroxysuccinimide
  • PBA hydrophobic phenylboronic acid
  • the derivatized insulin is made by:
  • R 9 carboxyphenylboronic acid group to form R 9 -(CH 2 ) q -CO-0-CH 3 , where q is an integer from 3-25 and R 9 is a phenylboronic acid group, or
  • derivatized insulins made by the methods provided herein.
  • the method of making derivatized insulin includes screening of different derivatized insulins to identify derivatized insulins useful for treating diabetes. For example, an agglomerate of a plurality of the derivatized insulin molecules can be tested for glucose-responsive release of the derivatized insulin from the agglomerate. Glucose-responsive release of the derivatized insulin from the agglomerate identifies the derivatized insulin as useful for treating diabetes. Also provided are derivatized insulins made by the method.
  • Figure IB is a diagram showing the binding equilibrium of
  • Figure 2 is a diagram of the structures of example of lipid-modified insulin derivatives, with a variable-length alkyl segment terminated in one of several PBAs.
  • FIG 3 is a diagram of an example of a bile acid insulin conjugate.
  • Bile acid cholic acid in this instance
  • B29 lysine residue is attached via the B29 lysine residue, and modified with between 1 and 3 variable PBAs.
  • Figure 4 is a diagram of an example of pseudolysine insulin conjugate.
  • amino acid lysine or other amine-containing R-group
  • An amino acid is installed between two tunable alkyl segments to modify length of the hydrophobic segment.
  • Figures 5a, 5b, and 5c are graphs of serum albumin chromatography showing native insulin (a), standard long-lasting insulin detemir (LA-C14) (b) and one of the glucose-responsive insulin variants (c).
  • native insulin a
  • LA-C14 standard long-lasting insulin detemir
  • c glucose-responsive insulin variants
  • Figure 6 is a graph the relative insulin activity of 4 PBA-modified insulin variants compared to insulin detemir (Ins-LA-C14), all of which have been modified via the B29 lysine of insulin. No significant change in activity was observed for any of the modified variants.
  • the order of the graph lines after 9 hours are, from top to bottom, native insulin and Ins-PBA-A, with the rest overlapping.
  • the order of the graph lines after 12 hours are, from top to bottom, native insulin, Ins-PBA-A, Ins-PBA-S, Ins-PBA-N, and Ins-PBA-F.
  • Figures 8a, 8b, 8c, and 8d are graphs of results from continuous glucose monitoring in mice treated with one PBA-modified insulin derivative (Ins-PBA- F) compared to a normal long-acting but non-glucose responsive version (Ins- LA-C14) as well as a healthy animal with a fully functioning pancreas, (a)
  • Figures 9a and 9b are graphs of blood glucose levels over time using insulin derivatives with an intraperitoneal tolerance test at 3 hours with glucose, fructose (a non-glucose diol), and PBS.
  • Figure 10 is a diagram of the structures of example derivatized insulins in Library 1.
  • FIGS 11 A and 1 IB are diagrams showing an example of the synthetic scheme for making example derivatized insulins in Library 1.
  • Figure 12 is a diagram of the structures of example derivatized insulins in Library 2.
  • Figures 13 A, 13B, and 13C are diagrams of the structures of example derivatized insulins in Library 3.
  • Figure 14 is a diagram of glucose-responsiveness of an example of derivatized insulin.
  • Figure 15 is a diagram of Scheme 1, Native human recombinant insulin was chemically modified on the B29 lysine.
  • Figures 16A-16F are diagrams of the chemical structures of azido diols and diol compounds, which were evaluated via n B NMR.
  • A 6-azido-6-deoxy- D-galactose
  • B 1 -Azido- l-deoxy-P-D-lactopyranoside
  • C a-D-
  • Figure 17 is a graph showing the affinity interaction of modified insulin with immobilized boronic acid resin.
  • the column was filled with 2 ml of boronic acid resin, loaded with 1 mg of insulin Analog.
  • the resin was washed with fresh PBS and the filtrate was collected as fractions. Insulin concentration was quantified using a micro-BSA kit with respect to Analog calibration curve.
  • Figure 18 is a graph showing n B NMR analysis for the stability of 4NBA:diol complexes at phosphate buffer, 0.1 M, pH 7.4, 37 ° C. Solid complexes were suspended in phosphate buffer saline and were agitated for 0, 0.05, 1 and 24 hours. Solids were then separated via centrifugation and freeze dried. Samples were dissolved in DMSO- d 6 at a concentration of 10 mM and were tested at room temperature.
  • Figures 19A and 19B are graphs showing hydrophobic PBAs (1TB A, 4DBA and 4NBA) tested as a function of glucose (A) and fructose (B) concentrations in PBS.
  • Figures 20A-20E are graphs showing dynamic light scattering
  • Figure 21 is a diagram of Scheme 2, Preparation of long-acting insulin formulations via hydrophobic interactions and PBA-diol interactions.
  • Reaction A DP3 -insulin is dissolved with 2 Zn 2+ per hexamer to induce self- association.
  • Reaction B DP3-Insulin Hexamers are conjugated with hydrophobic PBAs to generate self-associated insulin structures that have a hydrophobic outer layer.
  • Figures 22A-22C are graphs showing in vivo insulin data for SC injections of STZ induced mice.
  • A C12-insulin versus native insulin XI dose
  • B DP3 insulin versus DP3-insulin/C12-N-PBA versus native insulin XI dose
  • C DP3-insulin versus DP3-insulin/C12-N-PBA X 0.5 dose.
  • FIGS 23A-23G are diagrams of representative structures of derivatized insulin.
  • Core structures A-C have a phenylboronic acid (PBA) moiety, which could bind with glucose to change the properties of insulin.
  • Core structures D-F have a PBA group and a glucamine, which can self-associate to form aggregates.
  • the R group in red can be appended by different functional groups and substituents, shown in G, which provide the diversity of the whole library.
  • Figures 24A-24E are diagrams showing examples of monomer design, oligomer synthesis, and conjugation of oligomers to siRNA.
  • Amine, alcohol, and carboxylic acid moieties are used for monomer functionalization and controlled oligomerzation. Amine and alcohol moieties can be used for oligomerization, while carboxylic acid moieties are used for functionalization of the monomers,
  • Examples of delivery-relevant functionalities for monomer functionalization are used.
  • Representative structures of functionalized monomers (d) Synthetic strategy used in oligomeric synthesis, (e) Successful conlugation of oligomeric sequencesto dibenzocyclooctyne siRNA utilizing copper-free Huisgen cycloaddition.
  • Figure 25 is a diagram of alternative monomer backbone frameworks.
  • Figure 26 is a diagram of representative oligomeric structures for conjugation to insulin.
  • Controlling blood glucose levels refers to the maintenance of blood glucose concentrations at a desired level, typically between 70-130 mg/dL or 90- l lO mg/dL.
  • Dosage unit form refers to a physically discrete unit of conjugate appropriate for the patient to be treated.
  • Hydrophilic refers to molecules which have a greater affinity for, and thus solubility in, water as compared to organic solvents.
  • the hydrophilicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water- immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is present in the water than in the organic solvent, then the compound is considered hydrophilic.
  • Hydrophobic refers to molecules which have a greater affinity for, and thus solubility in, organic solvents as compared to water.
  • the hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water- immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is present in the organic solvent than in the water, then the compound is considered hydrophobic.
  • Peptide as used herein includes “polypeptide,” “oligopeptide,” and refers to a chain of at a-amino acid residues linked together by covalent bonds (e.g., peptide bonds). The length of the peptide is limited at the lower end only by the minimum number amino acids required to form a self-assembling peptide.
  • “Pharmaceutically acceptable carrier” as used herein means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or excipient.
  • oligomeric describes something made primarily from a plurality of monomeric units and is generally referred to as an "oligomer.”
  • An oligomer can have a molecular weight between 10 Daltons and 15,000 Daltons, between 100 Daltons and 10,000 Daltons, or between 500 Daltons and 5,000 Daltons.
  • An oligomer can have from 3 to 100 monomeric units, from 4 to 50 monomeric units, or from 5 to 25 monomeric units.
  • Biocompatible and biologically compatible generally refer to materials that are, along with any metabolites or degradation products thereof, generally non-toxic to the recipient, and do not cause any significant adverse effects to the recipient.
  • biocompatible materials are materials which do not elicit a significant inflammatory, immune or toxic response when administered to an individual.
  • smart delivery system or “interactive delivery system”, as used interchangeably herein, refer to a delivery system for one or more therapeutic, prophylactic, or diagnostic agents wherein the rate of delivery is responsive to one or more stimuli indicative of the need for delivery.
  • a smart insulin delivery system delivers insulin at a rate that is dependent upon the glucose levels in proximity to the delivery system.
  • the total response i.e. the total amount of insulin released or made biologically available will depend upon the total time the glucose level is high enough to promote dissociation, i.e., the amount of time it takes to restore normoglycemia.
  • the response is preferably pulsatile, and preferably little to no insulin is released at hypo- or normoglycemia.
  • the insulin derivatives described herein should have a lower rate of dissociation at normoglycemia than at hyperglycemia.
  • pulsatile or “pulsatile release,” as used herein, refers to the release of multiple doses from a single administration to a subject.
  • the individual doses can be administered at a variety of intervals, depending on the formulation of the delivery system and the application.
  • a smart pulsatile delivery system is capable of administering multiple doses of a therapeutic, prophylactic, or diagnostic agent in response to one or more stimuli, preferably wherein the dosage delivered is responsive to the deviation of the stimuli from a target value.
  • a smart pulsatile insulin delivery system preferably delivers little to no insulin during periods of normoglycemia but delivers a dosage of insulin in response to hypoglycemic conditions that is responsive to the deviation from normoglycemia, preferably in an amount sufficient to restore normglycemic glucose levels.
  • the amount of insulin derivative released depends upon the glucose level and the time to locally restore normglycemia; more generally, the amount released should depend upon the deviation of the external stimulus from the normal value and the time needed to return to the normal value.
  • an amino acid residue having a carboxylic acid group in the side chain designates amino acid residues like Asp, Glu and hGlu.
  • the amino acids can be in either the L- or D-configuration. If nothing is specified it is understood that the amino acid residue is in the L configuration.
  • an amino acid residue having a neutral side chain designates amino acid residues like Gly, Ala, Val, Leu, He, Phe, Pro, Ser, Thr, Cys, Met, Tyr, Asn and Gin.
  • activated acid is meant a carboxylic acid in which an activated leaving group has been attached to the acyl carbon enabling reaction with an amino group under formation of an amide bond and release of the leaving group.
  • Activated fatty acids may be activated esters of fatty acids, activated amides of fatty acids and anhydrides or chlorides.
  • Activated fatty acid includes derivatives thereof such as N-hydroxybenzotriazole and N-hydroxysuccinimide.
  • fatty acid is meant a linear or branched carboxylic acids having at least 2 carbon atoms and being saturated or unsaturated.
  • fatty acids are capric acid, lauric acid, tetradecanoic acid (myristic acid), pentadecanoic acid, palmitic acid, heptadecanoic acid, and stearic acid.
  • Alkyl refers to the radical of saturated or unsaturated aliphatic groups, including straight-chain alkyl, alkenyl, or alkynyl groups, branched-chain alkyl, alkenyl, or alkynyl groups, cycloalkyl, cycloalkenyl, or cycloalkynyl (alicyclic) groups, alkyl substituted cycloalkyl, cycloalkenyl, or cycloalkynyl groups, and cycloalkyl substituted alkyl, alkenyl, or alkynyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), more preferably 20 or fewer carbon atoms, more preferably 12 or fewer carbon atoms, and most preferably 8 or fewer carbon atoms.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • the ranges provided above are inclusive of all values between the minimum value and the maximum value.
  • alkyl includes both "unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having one or more substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents include, but are not limited to, halogen, hydroxyl, carbonyl (such as a carboxyl, alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety.
  • carbonyl such as a carboxyl, alkoxycarbonyl, formyl, or an acyl
  • thiocarbonyl such as a thioester, a
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths.
  • Preferred alkyl groups are lower alkyls.
  • the alkyl groups may also contain one or more heteroatoms within the carbon backbone.
  • the heteroatoms incorporated into the carbon backbone are oxygen, nitrogen, sulfur, and combinations thereof.
  • the alkyl group contains between one and four heteroatoms.
  • Alkenyl and Alkynyl refer to unsaturated aliphatic groups containing one or more double or triple bonds analogous in length (e.g., C 2 -C 30 and the preferred ranges discussed above) and possible substitution to the alkyl groups described above.
  • Aryl refers to 5-, 6- and 7-membered aromatic ring.
  • the ring may be a carbocyclic, heterocyclic, fused carbocyclic, fused
  • heterocyclic, bicarbocyclic, or biheterocyclic ring system optionally substituted by halogens, alkyl-, alkenyl-, and alkynyl-groups.
  • Ar includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "heteroaryl", “aryl heterocycles", or “heteroaromatics”.
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties,— CF 3 , --CN, or the like.
  • Ar also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
  • heterocyclic ring examples include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl,
  • benzoxazolyl benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-
  • Alkylaryl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or hetero aromatic group).
  • Heterocycle refers to a cyclic radical attached via a ring carbon or nitrogen of a monocyclic or bicyclic ring containing 3-10 ring atoms, and preferably from 5-6 ring atoms, consisting of carbon and one to four heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) wherein Y is absent or is H, O, (C 1-4 ) alkyl, phenyl or benzyl, and optionally containing one or more double or triple bonds, and optionally substituted with one or more substituents.
  • heterocycle also encompasses substituted and unsubstituted heteroaryl rings. Examples of heterocyclic ring include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
  • Heteroaryl refers to a monocyclic aromatic ring containing five or six ring atoms consisting of carbon and 1, 2, 3, or 4 heteroatoms each selected from the group consisting of non-peroxide oxygen, sulfur, and N(Y) where Y is absent or is H, O, (Ci-Cg) alkyl, phenyl or benzyl.
  • Non- limiting examples of heteroaryl groups include furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl, pyrimidinyl (or its N- oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide) and the like.
  • heteroaryl can include radicals of an ortho-fused bicyclic heterocycle of about eight to ten ring atoms derived therefrom, particularly a benz-derivative or one derived by fusing a propylene, trimethylene, or tetramethylene diradical thereto.
  • heteroaryl can be furyl, imidazolyl, triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl, pyraxolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl (or its N-oxide), thientyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or its N-oxide), quinolyl (or its N-oxide), and the like.
  • Halogen refers to fluorine, chlorine, bromine, or iodine.
  • substituted refers to all permissible substituents of the compounds described herein.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, but are not limited to, halogens, hydroxyl groups, or any other organic groupings containing any number of carbon atoms, preferably 1-14 carbon atoms, and optionally include one or more heteroatoms such as oxygen, sulfur, or nitrogen grouping in linear, branched, or cyclic structural formats.
  • substituents include alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, phenyl, substituted phenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, halo, hydroxyl, alkoxy, substituted alkoxy, phenoxy, substituted phenoxy, aroxy, substituted aroxy, alkylthio, substituted alkylthio, phenylthio, substituted phenylthio, arylthio, substituted arylthio, cyano, isocyano, substituted isocyano, carbonyl, substituted carbonyl, carboxyl, substituted carboxyl, amino, substituted amino, amido, substituted amido, sulfonyl, substituted sulfonyl, sulfonic acid, phosphoryl, substituted phosphoryl, phosphonyl, substituted phosphonyl, polyaryl
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. It is understood that “substitution” or “substituted” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e. a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • a compound When a compound is stated to be “soluble at physiological pH values” it means that the compound can be used for preparing compositions that are fully dissolved at physiological pH values. Such favorable solubility may either be due to the inherent properties of the compound alone or a result of a favorable interaction between the compound and one or more ingredients contained in the vehicle.
  • the insulin derivative includes a chemical group capable of binding to or reacting with glucose.
  • reversible glucose sensors are organic borates, preferably aryl boronates or other borates. Boronic acids covalently react with cis-diols to form five or six membered cyclic esters in an alkaline aqueous solution, which dissociates in acidic pH.
  • Boronate sensors that bind glucose under physiological conditions are preferred.
  • useful boronates include, but are not limited to, aryl boronates, aminomethyl-aryl-2-boronates, and other boronates with amino groups in the vicinity or aryl boronates substituted with electron- withdrawing groups for example, sulfo-, carboxy-, nitro-, cyano-, fluoro-phenyl boronates, pyridine boronates, pyridinium boronates or their combinations.
  • Diboronates may be employed to provide glucose selectivity over for instance fructose and lactate.
  • the hydrophilic domain of the self- assembling peptide is terminated with phenylboronic acid (PBA) group.
  • PBA phenylboronic acid
  • a PBA group is a compound, residue or moiety comprising a benzene with a boronic acid functional group.
  • the ring can be further substituted or not further substituted (beyond the boronic acid functional group and the covalent linkage to backbone structure) and can be amino heterocyclic.
  • a PBA group can have the structure:
  • Ri 6 is NH, NR29, or is not present
  • Ri 7 is CH 2 , CO, S0 2 , or is not present
  • Rig, Ri9, R 2 o, R 2 i, and R 22 are each independently -B(OH) 2 , -F, -N0 2 , -CN, -H, or not present, where one and only one of Ri 8 , R19, R 2 o, R 2 i, and R 22 is -B(OH) 2
  • R 2 3, R 2 4, R 2 5, R 2 6, and R 27 are each independently C or N, where at most only three of R 23 , R24, R 2 5, R 2 6, and R 27 are N
  • R29 is Ci_ 4 alkyl.
  • the PBA structure can be designed to bind glucose at a physiological value.
  • the pKa of traditional phenylboronic acid is approximately 8.9.
  • the PBA conjugate is chemically modified to lower the pK to less than 8.9 (Matsumoto A. et al, Chem. Commun. , 2010, 46, 2203-2205). Based on this value, only a limited percentage of PBA should be able to covalently bind glucose at physiological pH. However, there is sufficient glucose interaction to affect agglomeration of the compositions.
  • the phenylboronic acid moieties are in equilibrium between the charged (anionic) and uncharged form as shown in Figure IB. Only charged
  • phenylboronic acid moieties can form a stable complex with glucose.
  • the complex between the uncharged form and glucose is unstable because of its high susceptibility to hydrolysis.
  • the equilibrium is shifted in the direction of increasing charged phenylboronic acid groups.
  • the increasing negative charge on the PBA groups results in disruption of the secondary structure causing the compositions to dissociate and make therapeutic biologically available. Therefore, the rate of such therapeutic release adapts to glucose level fluctuations.
  • the amount of negatively charged PBA is increased with the increase of the glucose level, which results in the dissociation of the compositions and subsequent increase of the rate of the therapeutic release.
  • compositions described herein can be used for the responsive and/or controlled delivery of one or more therapeutic, prophylactic, or diagnostic agents.
  • compositions contain only a single
  • therapeutic, prophylactic, or diagnostic agent i.e. insulin.
  • multiple agents can be delivered either in a responsive manner or in a controlled manner, either together or independently.
  • therapeutic, prophylactic, or diagnostic agents include insulin, insulin analogs, glucagon, GLP-1, or a GLP-1 agonist can be used.
  • Combinations of derivatives of different therapeutic, prophylactic, or diagnostic agents can be used together in compositions for treating subjects.
  • compositions are provided containing insulin or an insulin analog.
  • Insulin refers to a natural peptide hormone made by the pancreas that controls the level of the sugar glucose in the blood. Insulin permits cells to use glucose.
  • Human insulin has three primary amino groups: the N- terminal group of the A-chain and of the B-chain and the ⁇ -amino group of LysB29. Any of these primary amines, or a primary amine added in an insulin analog, can be used to as the attachment point of derivatizing molecules and groups.
  • Insulin analog refers to human insulin in which one or more amino acid residues have been replaced by another amino acid residue or deleted or in which the A chain and/or the B chain has been extended by addition of one or more amino acid residues at the N-terminal or at the C- terminal and which controls the level of glucose in the blood but with different pharmacokinetics than the naturally occurring insulin.
  • insulin analogs include NPH insulin; also known as Humulin N, Novolin N, Novolin NPH, NPH Iletin II, and isophane insulin, marketed by Eli Lilly and Company under the name Humulin N, is an intermediate-acting insulin given to help manage the blood sugar level of those with diabetes. Many people reported problems following being switched to these insulins in the 80s, from
  • insulin derivative as used herein is meant a naturally occurring insulin or an insulin analogue which has been chemically modified, e.g., by introducing a side chain in one or more positions of the insulin backbone or by oxidizing or reducing groups of the amino acid residues in the insulin or by acylating a free amino group or a hydroxy group.
  • desB30 or “B(l-29)” is meant a natural insulin B chain or an insulin analog thereof lacking the B30 amino acid residue and by "A(l-21)” is meant the natural insulin A chain or an analog thereof.
  • DesB30,desB29 human insulin is a human insulin lacking B29 and B30.
  • Bl amino acid residue in position 1 in the B chain of insulin (counted from the N-terminal end) and the amino acid residue in position 1 in the A chain of insulin (counted from the N-terminal end), respectively.
  • the amino acid residue in a specific position can also be denoted as, e.g., Phe B1 , which means that the amino acid residue in position Bl is a phenylalanine residue.
  • the insulin analogs can be such that position 28 of the B chain can be modified from the natural Pro residue to Asp, Lys, or He. Lys in position B29 can also be modified to Pro. Furthermore B30 can be Lys in which case B29 is different from Cys, Met, Arg and Lys.
  • Asn at position A21 can be modified to Ala, Gin, Glu, Gly, His, He, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and in particular to Gly.
  • Asn at position B3 can be modified to Lys or Asp.
  • Further examples of insulin analogs are des(B30) human insulin, insulin analogs where one or both of Bl and B2 have been deleted; insulin analogs where the A-chain and/or the B-chain have an N-terminal extension and insulin analogs where the A-chain and/or the B-chain have a C-terminal extension. Further insulin analogs are such that one or more of B26-B30 have been deleted.
  • Lispro Eli Lilly and Company had the first insulin analog with "lispro" as a rapid acting insulin analog. It is marketed under the trade name Humalog. It was engineered through recombinant DNA technology so that the penultimate lysine and proline residues on the C-terminal end of the B-chain were reversed. This modification did not alter the insulin receptor binding, but blocked the formation of insulin dimers and hexamers. This allowed larger amounts of active monomeric insulin to be available for postprandial (after meal) injections.
  • NovoLog/NovoRapid (UK-CAN) as a rapid acting insulin analog. It was created through recombinant DNA technology so that the amino acid, B28, which is normally proline, is substituted with an aspartic acid residue. The sequence was inserted into the yeast genome, and the yeast expressed the insulin analog, which was then harvested from a bioreactor. This analogue also prevents the formation of hexamers, to create a faster acting insulin. It is approved for use in CSII pumps and Flexpen, Novopen delivery devices for subcutaneous injection.
  • Glulisine is a newer rapid acting insulin analog from Sanofi- Aventis, approved for use with a regular syringe, in an insulin pump or the Opticlik Pen. Standard syringe delivery is also an option. It is sold under the name Apidra. The FDA-approved label states that it differs from regular human insulin by its rapid onset and shorter duration of action. Shifted isoelectric point insulins. Normal unmodified insulin is soluble at physiological pH. Analogues have been created that have a shifted isoelectric point so that they exist in a solubility equilibrium in which most precipitates out but slowly dissolves in the bloodstream and is eventually excreted by the kidneys.
  • insulin analogs and derivatives are used to replace the basal level of insulin, and may be effective over a period of up to 24 hours.
  • some insulin derivatives such as insulin detemir, bind to albumin rather than fat like earlier insulin varieties, and results from long-term usage (e.g. more than 10 years) have never been released.
  • Glargine insulin Glargine insulin.
  • Sanofi-Aventis developed glargine as a longer lasting insulin analog, and markets it under the trade name Lantus. It was created by modifying three amino acids. Two positively charged arginine molecules were added to the C-terminus of the B-chain, and they shift the isoelectric point from 5.4 to 6.7, making glargine more soluble at a slightly acidic pH and less soluble at a physiological pH. Replacing the acid-sensitive asparagine at position 21 in the A-chain by glycine is needed to avoid deamination and dimerization of the arginine residue. These three structural changes and formulation with zinc result in a prolonged action when compared with biosynthetic human insulin.
  • Detemir insulin Novo Nordisk created insulin detemir and markets it under the trade name Levemir as a long-lasting insulin derivative for
  • the basal level of insulin may be maintained for up to 20 hours, but the time is clearly affected by the size of the injected dose. This insulin has a high affinity for serum albumin, increasing its duration of action. 2. Diabetes Medications
  • Exemplary diabetes medications include sulfonylureas, meglitinides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, or DPP-4 inhibitors.
  • Sulfonylureas stimulate the beta cells of the pancreas to release more insulin.
  • Chlorpropamide (Diabinese) is the only first-generation sulfonylurea still in use today.
  • the second generation sulfonylureas are used in smaller doses than the first-generation drugs. There are three second-generation drugs:
  • glipizide Glucotrol and Glucotrol XL
  • glyburide Micronase, Glynase, and Diabeta
  • glimepiride Amaryl
  • Meglitinides are drugs that also stimulate the beta cells to release insulin.
  • Repaglinide Prandin
  • nateglinide Starlix
  • Metformin Glucophage
  • Biguanides lower blood glucose levels primarily by decreasing the amount of glucose produced by the liver.
  • Rosiglitazone Avandia
  • pioglitazone ACTOS
  • DPP-4 inhibitors help improve AIC without causing hypoglycemia. They work by preventing the breakdown of a naturally occurring compound in the body, GLP-1. GLP-1 reduces blood glucose levels in the body, but is broken down very quickly so it does not work well when injected as a drug itself. By interfering in the process that breaks down GLP-1, DPP-4 inhibitors allow it to remain active in the body longer, lowering blood glucose levels only when they are elevated.
  • Sitagliptin JANUVIA
  • saxagliptin ONGLYZA
  • compositions are provided containing an insulin derivative and one or more additional diabetes medications that can be delivered together in a responsive manner, or independently by providing extended release of the diabetes medication in combination with responsive release of the insulin derivative in response to increased glucose levels.
  • other therapeutic, prophylactic or diagnostic agents can be encapsulated to treat or manage diseases or disorders.
  • These can include small drugs, proteins or peptide, nucleic acid molecules such as DNA, mR A and siR A, polysaccharides, lipids, and combinations thereof.
  • compositions containing a polymeric matrix responsive to blood alcohol levels it may be advantageous to use one or more drugs commonly used for treating alcoholism or other addictions, i.e. disulfiram or calcium carbamide, diazepam or librium, or an opiate antagonists such as naloxone, naltrexone, cyclazocine,
  • diprenorphine diprenorphine, etazocine, levalorphan, metazocine, or nalorphine.
  • Diagnostic agents may be release alone or in combination with therapeutic and/or prophylactic agents. Examples include radionuclides, radiopaque molecules, and MRI, x-ray or ultrasound detectable molecules.
  • compositions containing an insulin derivative may be administered parenterally to subjects in need of such a treatment.
  • Parenteral administration can be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe.
  • parenteral administration can be performed by means of an infusion pump.
  • Further options are to administer the insulin nasally or pulmonally, preferably in compositions, powders or liquids, specifically designed for the purpose.
  • Injectable compositions of the insulin derivatives can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product.
  • an insulin derivative can be dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared.
  • An isotonic agent, a preservative and a buffer can be added as required and the pH value of the solution is adjusted— if necessary— using an acid, e.g., hydrochloric acid, or a base, e.g., aqueous sodium hydroxide, as needed.
  • the volume of the solution can be adjusted with water to give the desired concentration of the ingredients.
  • the buffer can be selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof.
  • the formulation can further comprise a pharmaceutically acceptable preservative which can be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2- phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3p- chlorphenoxypropane-l,2-diol) or mixtures thereof.
  • a pharmaceutically acceptable preservative which can be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phen
  • the preservative can be present in a concentration from 0.1 mg/ml to 20 mg/ml. In some embodiments, the preservative can be present in a concentration from 0.1 mg/ml to 5 mg/ml. In some embodiments, the preservative can be present in a concentration from 5 mg/ml to 10 mg/ml. In some embodiments, the
  • preservative can be present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives and their combinations constitutes an alternative embodiment.
  • compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, current edition.
  • the formulation can further comprise an isotonic agent which can be selected from the group consisting of a salt (e.g., sodium chloride), a sugar or sugar alcohol, an amino acid (e.g., glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g., glycerol (glycerine), 1 ,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3- butanediol) polyethyleneglycol (e.g., PEG400), or mixtures thereof.
  • a salt e.g., sodium chloride
  • a sugar or sugar alcohol e.g., an amino acid (e.g., glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, thre
  • Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used.
  • sugars that readily complex with phenylboronic acid can be avoided in compositions using insulin derivatives with a phenylboronic acid group.
  • the sugar additive can be sucrose.
  • Sugar alcohol is defined as a C 4 -C 8 hydrocarbon having at least one -OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol.
  • the sugar alcohol additive can be mannitol.
  • the sugars or sugar alcohols mentioned above can be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid preparation and does not adversely affect the effects achieved using the insulin derivatives (such as glucose-responsiveness).
  • the sugar or sugar alcohol concentration can be between about 1 mg/ml and about 150 mg/ml.
  • the isotonic agent can be present in a
  • the isotonic agent can be present in a concentration from 1 mg/ml to 50 mg/ml. In some embodiments, the isotonic agent can be present in a concentration from 1 mg/ml to 7 mg/ml. In some embodiments, the isotonic agent can be present in a concentration from 8 mg/ml to 24 mg/ml. In some embodiments, the isotonic agent can be present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents and their combinations constitutes an alternative embodiment. The use of an isotonic agent in pharmaceutical compositions is well-known.
  • Typical isotonic agents are sodium chloride, mannitol, dimethyl sulfone and glycerol and typical preservatives are phenol, m-cresol, methyl p- hydroxybenzoate and benzyl alcohol.
  • suitable buffers are sodium acetate, glycylglycine, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) and sodium phosphate.
  • a composition for nasal administration of an insulin derivative can, for example, be prepared as described in European Patent No. 272097 (to Novo Nordisk A/S).
  • Compositions containing insulin derivatives can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycaemia, for example, as sometimes seen in seriously injured persons and persons who have undergone major surgery.
  • the optimal dose level for any subject will depend on a variety of factors including the efficacy of the specific insulin derivative employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily or periodic dosage of the insulin derivative of this invention be determined for each individual subject by those skilled in the art in a similar way as for known insulin compositions.
  • the insulin derivatives can be used in mixture with other types of insulin, e.g., insulin analogs with a more rapid onset of action.
  • insulin analogs e.g., insulin analogs with a more rapid onset of action. Examples of such insulin analogs are described, e.g., in the European patent applications having the publication Nos. EP 214826 (Novo Nordisk A/S), EP 375437 (Novo Nordisk A/S) and EP 383472 (Eli Lilly & Co.).
  • the present compounds can be administered in combination with one or more further active substances in any suitable ratios.
  • Such further active agents can be selected from antidiabetic agents,
  • Suitable antidiabetic agents include insulin, GLP-1 (glucagon like peptide-1) derivatives such as those disclosed in WO 98/08871 (Novo Nordisk A/S), which is incorporated herein by reference, as well as orally active hypoglycemic agents.
  • Suitable orally active hypoglycemic agents preferably include imidazolines, sulfonylureas, biguanides, meglitinides, oxadiazolidinediones, thiazolidinediones, insulin sensitizers, a-glucosidase inhibitors, agents acting on the ATP-dependent potassium channel of the pancreatic ⁇ -cells, e.g., potassium channel openers such as those disclosed in WO 97/26265, WO 99/03861 and WO 00/37474 (Novo Nordisk A/S) which are incorporated herein by reference, potassium channel openers, such as ormitiglinide, potassium channel blockers such as nateglinide or BTS-67582, glucagon antagonists such as those disclosed in WO 99/01423 and WO 00/39088 (Novo Nordisk A S and Agouron
  • GLP-1 agonists such as those disclosed in WO 00/42026 (Novo Nordisk A/S and Agouron Pharmaceuticals, Inc.), which are incorporated herein by reference, DPP-IV (dipeptidyl peptidase-IV) inhibitors, PTPase (protein tyrosine phosphatase) inhibitors, inhibitors of hepatic enzymes involved in stimulation of gluconeogenesis and/or glycogenolysis, glucose uptake modulators, GSK-3 (glycogen synthase kinase-3) inhibitors, compounds modifying the lipid metabolism such as antihyperlipidemic agents and antilipidemic agents, compounds lowering food intake, and PPAR (peroxisome proliferator-activated receptor) and RXR (retinoid X receptor) agonists such as ALRT-268, LG-1268 or LG-1069.
  • DPP-IV dipeptidyl peptidase-IV
  • PTPase protein tyrosine phosphatase
  • Insulin derivatives can be provided in the form of essentially zinc free compounds or in the form of zinc complexes.
  • zinc complexes of an insulin derivative When zinc complexes of an insulin derivative are provided, two Zn 2+ ions, three Zn 2+ ions or four Zn 2+ ions can be bound to each insulin hexamer. Solutions of zinc complexes of the insulin derivatives will contain mixtures of such species.
  • a pharmaceutical composition comprising a therapeutically effective amount of an insulin derivative together with a pharmaceutically acceptable carrier can be provided for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a subject in need of such a treatment.
  • An insulin derivative can be used for the manufacture of a pharmaceutical composition for use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.
  • a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a subject in need of such a treatment comprising a therapeutically effective amount of an insulin derivative in mixture with an insulin or an insulin analog which has a rapid onset of action, together with pharmaceutically acceptable carriers and additives.
  • an insulin derivative which is soluble at physiological pH values is provided. In some embodiments, an insulin derivative according to the invention which is soluble at pH values in the interval from about 6.5 to about 8.5 is provided. In some embodiments, an insulin derivative which binds serum albumin is provided. In some embodiments, an insulin derivative which forms insulin hexamers is provided. In some embodiments, an insulin derivative which agglomerates is provided. In some embodiments, an insulin derivative which forms conjugates is provided.
  • a pharmaceutical composition comprising an insulin derivative where the insulin derivative binds serum albumin is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative where the insulin derivative forms insulin hexamers is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative where the insulin derivative agglomerates is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative where the insulin derivative forms conjugates is provided.
  • a pharmaceutical composition comprising an insulin derivative which is soluble at physiological pH values is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative according to the invention which is soluble at pH values in the interval from about 6.5 to about 8.5 is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative which binds serum albumin is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative which forms insulin hexamers is provided. In some embodiments, a pharmaceutical composition comprising an insulin derivative which agglomerates is provided. In some embodiments, a
  • composition comprising an insulin derivative which forms conjugates.
  • a pharmaceutical composition with a prolonged profile of action which comprises an insulin derivative is provided.
  • a pharmaceutical composition which is a solution containing from about 50 U/ml to about 1000 U/ml, from about 200 U/ml to about 1000 U/ml, from about 200 U/ml to about 500 U/ml, from about 300 U/ml to about 1000 U/ml, or from about 300 U/ml to about 500 U/ml, of an insulin derivative or of a mixture of the insulin derivative with a rapid acting insulin analog is provided.
  • One unit of native insulin is approximately 36 ⁇ g of insulin. Equivalent units for insulin derivatives and analogs will vary from this and can be determined by those of skill in the art.
  • a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 2400 nmol/ml, from about 400 nmol/ml to about 1200 nmol/ml, from about 600 nmol/ml to about 2400 nmol/ml, or from about 600 nmol/ml to about 1200 nmol/ml of an insulin derivative or of a mixture of the insulin derivative with a rapid acting insulin analog is provided.
  • Insulin-binding peptides for facilitating glucose-dependent insulin activity and/or solubility are also provided.
  • a combinatorial library of peptides which incorporate amino acids with glucose-responsive moieties, such as phenylboronic acid groups can be used to identify peptides that bind insulin and that, through that binding, affect activity and/or solubility of the insulin. Such effects of identified peptides can be used to alter bioavailability of insulin based on glucose concentration. Screening of the peptides can be accomplished by, for example, exposing immobilized insulin to the complete peptide library and washing with detergent to elute non-binding or weakly binding compounds.
  • the immobilized insulin can be washed with varying concentrations of glucose and the eluted peptides collected as peptides with glucose-responsive insulin binding.
  • This method allows rapid screening of a huge library on a manageable scale and will produce a subset of hits that are insulin binding.
  • the eluent containing the presumed insulin-binding sequences will be identified using mass spectrometry (MS) techniques. As it is assumed that the sample will be complex, peptides will be separated by a reversed-phase HPLC column in-line with the MS, and tandem MS/MS will be used to determine the peptide sequence. In order to improve the certainty of identification of hits, the eluent will be split into several fractions for independent sampling, and the same library will be screened 10 times by the protocol in 2, and hits will be ranked by the number of times that they appear in each of these screens.
  • MS mass spectrometry
  • the resultant peptide hits from MS screening will be resynthesized by solid phase techniques. These peptides hits will be separately incubated with insulin, and this insulin complex will be subsequently tested for insulin receptor activation when compared to native insulin in a cell-based assay.
  • the envisioned assay will measure the level of phosphorylated Akt, a signaling intermediate downstream of the activated insulin receptor.
  • insulin activity will be monitored in the presence of the peptide hit and hyperglycemic levels of glucose. This will validate whether suppression of insulin activity by the hit peptides is glucose-responsive.
  • the formulation of delivery of insulin with peptide hits will be examined to determine whether a more efficient glucose-responsive trigger can be generated.
  • protein engineering we can explore tethering the peptide to insulin using a flexible linker to improve the binding and responsiveness of the compound.
  • multivalent peptide structures can be explored to evaluate whether the formation of higher order insulin-peptide aggregates could be used to improve the delivery and the potency of the therapy.
  • the insulin- peptide complexes will also be formulated with existing drug delivery platforms, for example polymeric nanoparticles, to optimize drug delivery kinetics and protect both the insulin and peptide from enzymatic degradation.
  • the insulin/peptide formulation will be injected subcutaneously or intravenously into streptazotocin-induced diabetic mice.
  • the blood glucose of these mice will be monitored to determine the efficacy and duration of glycemic correction, including probing the insulin-responsiveness by bolus glucose challenge of reversed mice, and monitoring for the appropriate dose to ensure long-term normoglycemia.
  • blood glucose will be monitored every 30 minutes after injection until 8 hours.
  • blood glucose will be monitored 4 times a day for 2 weeks. Promising candidates will be evaluated in additional models with help from Sanofi for both safety and efficacy.
  • Insulin or insulin analogs can be derivatized using any suitable techniques.
  • the starting product for the acylation of the parent insulin or insulin analog or a precursor thereof can be produced by either well-known organic synthesis or by well-known recombinant production in suitable transformed microorganisms.
  • the insulin starting product can be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture.
  • desB(30) human insulin can be produced from a human insulin precursor B(l-29)-Ala- Ala-Lys-A(l-21) which is produced in yeast as disclosed in U.S. Pat. No.
  • This insulin precursor can then be converted into desB30 human insulin by ALP cleavage of the Ala-Ala-Lys peptide chain to give desB30 human insulin which can then be acylated to give the present insulin derivatives.
  • Dosage forms may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising known excipients and auxiliaries which facilitate processing into preparations which can be used pharmaceutically.
  • the formulation prior to injection, is in the form of a suspension.
  • the formulation is an injectable formulation.
  • An injectable insulin formulation can be made by suspending the insulin derivative in a diluent. The suspension is sterilized and filled in a vial suitable for unit or multiple injection dosing.
  • Sterile injectable preparations may be formulated as known in the art. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution.
  • the injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Components and compositions for such formulations are described further elsewhere herein.
  • the formulations can be administered subcutanteously, intramuscularly, or intradermally.
  • the formulation is injected subcutaneously.
  • a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a subject in need of such a treatment comprising administering to the subject a therapeutically effective amount of an insulin derivative together with a pharmaceutically acceptable carrier and pharmaceutical acceptable additives.
  • a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a subject in need of such a treatment comprising administering to the subject a therapeutically effective amount of an insulin derivative in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier and pharmaceutical acceptable additives.
  • an insulin derivative for the manufacture of a medicament for blood glucose lowering. In some embodiments, there is provided a use of an insulin derivative for the
  • Dosage unit form refers to a physically discrete unit of conjugate appropriate for the patient to be treated.
  • the formulation is an insulin formulation designed to release insulin into systemic circulation over time with a basal release profile following injection in a patient.
  • the formulation is designed to release insulin into systemic circulation over time with a non-basal release profile following injection in a patient.
  • Exemplary non-basal release profiles include a regular human insulin release profile and a prandial release profile.
  • the formulation is designed to release insulin into systemic circulation over time with a regular human insulin release profile following injection in a patient.
  • the formulation is designed to release insulin into the systemic circulation over time with a prandial release profile following injection in a patient.
  • compositions and formulations including a responsive composition can be administered to a subject in need of delivery of a therapeutic
  • the patient is in need of administration of a therapeutic agent in response to increases in blood glucose levels, i.e., due to diabetes.
  • the glucose complexes with a glucose-sensing component such as phenylboronic acid (PBA).
  • PBA phenylboronic acid
  • the complexation of glucose alters the chemical and/or physical properties of the insulin derivative such that release of the insulin is facilitated.
  • insulin derivatives can bind serum albumin (thus keeping the insulin from being bioavailable) based on the chemical and/or physical properties of the insulin derivative.
  • the properties of the insulin derivative changes (such as by increasing the aqueous solubility of the insulin derivative), thus facilitating release of the insulin derivative from the albumin.
  • the insulin formulation is administered to patients who are not fully insulin dependent.
  • the formulation provides a sufficient amount of insulin to the patient during the day so that the patient does not require additional insulin-containing formulations to maintain his/her blood glucose levels within a safe range.
  • the patient is typically not fully insulin dependent.
  • the formulation is administered to a patient who is receiving intensive insulin therapy as one of the insulin-containing formulations administered to the patient during the day.
  • the formulation delivers insulin to the patient with a basal release profile.
  • controlling blood glucose levels refers to the maintenance of blood glucose concentrations at a desired level, typically between 70-130 mg/dL or 90-110 mg/dL.
  • the formulation when administered to a patient with diabetes is able to maintain normoglycemia (normal glycemic levels) for a period of up to 2 days, 5 days, 1 week, 2 weeks, one month, or up to two months.
  • This example demonstrates the synthesis and development of several classes of long-acting insulin derivatives that have been designed to afford glucose-mediated binding to serum albumin to prepare glucose-responsive insulin. This was achieved by combining a lipidic or hydrophobic moiety to facilitate binding of serum albumin (or other proteins, such as globulin, lipoprotein complexes-HDL, LDL, etc.) with a phenylboronic acid (PBA) moiety, a class of molecules known to bind to glucose and other cis-1,2 or cis- 1,3 diols. PBA compounds and PBA-containing polymers have previously demonstrated utility in glucose sensing and insulin delivery. However, direct conjugation of PBA to insulin has not yet been demonstrated to afford glucose- mediated control of insulin activity.
  • PBA phenylboronic acid
  • the insulin derivatives described in this example represent three separate libraries of modified insulins. All insulin derivative in this example are modified via the B29 lysine to contain a hydrophobic component as well as a
  • PBA phenylboronic acid
  • Figure 1 illustrates some representative PBA structures that can be incorporated into the design of these modified insulins. PBAs useful for this purpose can take many forms. Four structures were explored in the design of the molecules described in this example, but molecular design can be broadened to include any of a number of PBA chemistries, as illustrated in Figure 1.
  • Example structures for the first library of modified insulins are shown in Figure 2.
  • the full library of synthetized compounds can be found in Library 1.
  • This insulin library consists of modification of the B29 lysine on insulin via an alkyl segment terminated with a phenylboronic acid.
  • Hydrophobic moieties, such as alkyl segments are known to interact with hydrophobic domains of serum albumin, and form the basis for the effect observed by long-lasting insulin (detemir).
  • This general scheme is illustrated in Figure 2. This modular approach affords control over the length of the alkyl segment and control over the type of PBA used, including any of the structures shown in Figure 1.
  • FIG. 3 An example structure for the second library of modified insulins are shown in Figure 3.
  • the full library of synthetized compounds can be found in Library 2.
  • These insulins are modified with a bile acid conjugated to between 1 and 3 PBAs.
  • Bile acids including cholic acid, lithocholic acid, hyocholic acid, deoxycholic acid, hyodeoxycholic acid, and chenodeoxycholic acid, can be used as a core for these structures.
  • Bile acids are known to be strong binders to serum albumin, to afford the insulin with long-lasting properties.
  • These bile acids can be modified at hydroxyl groups located at several different positions within the structure. As with the past library, any of a number of PBAs could be added at these positions.
  • bile acid conjugates could have between 1-3 PBAs attached to the central bile acid core, with any PBA from the examples shown in Figure 1 being suitable.
  • the PBAs afford glucose sensing properties. This is illustrated in Figure 3, with the example showing a cholic acid core modified at 3 sites with a phenylboronic acid, attached via an ethanolamine spacer. This spacer length is, similarly variable.
  • FIG. 4 An example of a pseudolysine-modified insulin library is shown in Figure 4.
  • the full library of synthetized compounds can be found in Library 3.
  • This library combines a PBA off of a lysine residue.
  • a similar amine-containing amino acid could also be used, such as ornithine or aminopropanoic acid.
  • Any of the PBAs shown in Figure 1 could be used to modify this amine and provide the ability to bind to glucose.
  • This PBA-containing amino acid is flanked by 2 alkyl segments of variable length to facilitate binding with serum albumin.
  • the small molecule is again attached to the B29 lysine of insulin.
  • Long-lasting glucose-responsive insulins of the type proposed here are designed for the ability to undergo glucose-mediated binding to serum albumin.
  • chromatography with a serum albumin solid support can inform glucose-dependent binding to albumin.
  • An example of this is shown in Figure 5.
  • the shift in retention as the glucose concentration of the mobile phase suggests a glucose-mediated decrease in binding affinity for the chemically modified insulin (in this case Ins-PBA-F).
  • Insulins of this type may be useful as both long-acting and glucose-responsive insulin derivatives.
  • Chemically modifying insulin has the potential to inhibit the normal function of the protein. It is speculated that modification specifically via the B29-lysine amine preserves insulin function.
  • each of the PBA-modified insulin derivatives restored blood glucose to a normogylcemic level, whereas native insulin failed to reduce blood sugar.
  • Ins-PBA-S, Ins-PBA-F, and Ins-PBA-N derivatives reversed blood glucose to pre-challenge levels.
  • a second IPGTT performed 7 hours following insulin administration demonstrated that Ins-PBA-S, Ins-PBA-F, and Ins-PBA-N were again able to restore normoglycemic levels.
  • Ins-PBA-A was not able to restore normoglycemia following the second challenge.
  • a third IPGTT performed at 10 hours following insulin administration revealed that Ins- PBA-F and Ins-PBA-N were still able to restore normoglycemia, and Ins-PBA-F was especially potent in reversing blood glucose levels to pre-challenge values.
  • 4-Carboxy-3- fluorophenylboronic acid was purchased from Optima Chemical (Douglas, GA, USA).
  • O-(Benzotriazol- 1 -yl)-N,N,N',A/ -tetramethyluronium tetrafluoroborate (TBTU) was purchased from AnaSpec (Fremont, CA, USA).
  • Methyl 12-aminododecanoate 6 (5.08g, 22.19mmole) and 4- bromosulfonyl chloride (4.73g, 18.49mmole) were dissolved in dichloromethane (100ml) with triethylamine (7.73ml, 55.47mmole). The reaction was stirred overnight and the solvent was removed in vacuo. The reaction was then dissolved in ethyl acetate and extracted with water and brine. The organic layer was dried with MgS0 4 and evaporated in vacuo. Column chromatography (0- 20% MeOH in DCM) gave a white product (6.2g, 13.84mmole, 75%).
  • Methyl 12-aminododecanoate 6 (2.00 g, 8.73mmole), 4- carboxyphenylboronic acid (2.18g, 13.10mmole), and TBTU (4.2 lg,
  • Methyl 12-aminododecanoate 6 (2.00 g, 8.73mmole), 4-Carboxy-3- fluorophenylboronic acid (2.4 lg, 13.10mmole), and TBTU (4.2 lg,
  • Methyl 12-aminododecanoate 6 (2.00 g, 8.73mmole), 3-Carboxy-5- nitrophenylboronic acid (2.78g, 13.10mmole), and TBTU (4.2 lg, 13.10mmole), were dissolved in a 1 : 1 DMF/pyridine solution (100ml). The reaction was stirred overnight under nitrogen and the solvent was removed in vacuo. The reaction was dissolved in ethyl acetate and extracted with 30% citric acid and brine. The organic layer was dried with MgS0 4 and evaporated in vacuo. Column chromatography (0-20%> MeOH in DCM) gave a pale yellow product (3.18g, 7.53mmole, 85%).
  • Methyl 12-((4-bromophenyl)sulfonamido)dodecanoate 7 (2.96g, 6.61mmole), bis(pinacolato)diboron (2.52g, 9.91mmole), Pd(dppf)Cl 2 DCM (0.8 lg, 0.9mmole), and potassium acetate (2.60g, 26.43mmole) were dissolved in dioxane (100ml) and refluxed under nitrogen overnight. The reaction was filtered through Celite and the solvent removed in vacuo. The reaction was dissolved in ethyl acetate and extracted with water and brine. The organic layer was dried with MgS0 4 and evaporated in vacuo. Column chromatography (0- 20% MeOH in DCM) gave a white product (2.7 lg, 5.47mmole, 85%).
  • This example relates to combinatorial generation of a library of chemically modified human insulins for glucose responsive delivery.
  • a library of chemically modified, human insulins will be developed, with bioavailability that is dependent on local glucose concentration. Specifically:
  • Modified insulin analogs will be assayed for glucose-responsive aggregation in physiological pH using size-exclusive chromatography (SEC). Lead glucose-responsive modified insulin analogs will be scaled up evaluated for ability to bind to insulin receptor and activate the insulin signaling pathway.
  • SEC size-exclusive chromatography
  • Formulations of modified insulin analogs as aggregated particles will be generated to determine glucose-responsive insulin release in varying glucose levels in vitro. This study tests the insulin-releasing profile as a function of time and glucose concentrations.
  • Leads will be tested for glucose correction in diabetic mouse models. Responsiveness will be evaluated by monitoring insulin levels in response to glucose challenge. Promising candidates will be evaluated in additional models with Sanofi help for both safety and efficacy.
  • This example also relates to non-covalent insulin-binding peptides for glucose-dependent activity will be developed to facilitate a glucose trigger for insulin activity and/or solubility of insulin. Specifically:
  • the derivatization sstructures can be -CO-(CH 2 ) j -NH-CO-CRiR 2 , where j is an integer from 3-25, where Ri is -NH-R 12 or -NH-CO-CH 2 -CH 2 -CNRi 2 - R 32 , where R 32 is glucamine, gluconic acid, glucosamine, fructosamine, galactosamine, mannosamine, or other hexosamines; Ri 2 is selected from the group consisting of hydrogen,-S0 2 alkyl, -S0 2 cycloalkyl, -S0 2 heterocycloalkyl, -S0 2 aryl, -S0 2 heteroaryl, -COalkyl, -COcycloalkyl, -COheterocycloalkyl, - COaryl, -COheteroaryl, -CONHalkyl, -CONHcycloalkyl, -CONHcycloal
  • R 2 is -(CH 2 ) n -Rn, where n is an integer from 3-25; and Rn is a phenylboronic acid group.
  • n is 4.
  • Ri 2 is a sulfonyl chloride, isocyanate, carboxylic acid chloride, aldehyde, or hydrogen. Examples of structures are shown in Figure 23.
  • All synthesized modified insulin analogs will be first formulated with zinc(II), phenol, m-cresol and sodium chloride to induce aggregation. SEC will be used to measure the molecular weight of the insulin aggregate in the presence and absence of glucose in the mobile phase. Analogs with decreasing MW in the presence of glucose will be selected for scale-up synthesis and further characterizations .
  • Selected insulin analogs will be tested for their affinities to insulin receptor compared to native insulin.
  • An insulin activity assay will be performed to evaluate the ability of modified insulin to activate the insulin signaling pathway by measuring the level of phosphorylated Akt.
  • Immobilized insulin will be exposed to the complete peptide library, with subsequent detergent washes to elute non-binding or weakly binding compounds. Subsequently, elution will be performed using varying
  • Insulin-binding peptides for facilitating glucose-dependent insulin activity and/or solubility are also provided.
  • a combinatorial library of peptides which incorporate amino acids with glucose-responsive moieties, such as phenylboronic acid groups can be used to identify peptides that bind insulin and that, through that binding, affect activity and/or solubility of the insulin. Such effects of identified peptides can be used to alter bioavailability of insulin based on glucose concentration. Screening of the peptides can be accomplished by, for example, exposing immobilized insulin to the complete peptide library and washing with detergent to elute non-binding or weakly binding compounds.
  • the immobilized insulin can be washed with varying concentrations of glucose and the eluted peptides collected as peptides with glucose-responsive insulin binding. This method allows rapid screening of a huge library on a manageable scale and will produce a subset of hits that are insulin binding.
  • the eluent containing the presumed insulin-binding sequences will be identified using mass spectrometry (MS) techniques. As it is assumed that the sample will be complex, peptides will be separated by a reversed-phase HPLC column in-line with the MS, and tandem MS/MS will be used to determine the peptide sequence. In order to improve the certainty of identification of hits, the eluent will be split into several fractions for independent sampling, and the same library will be screened 10 times by the protocol in 2, and hits will be ranked by the number of times that they appear in each of these screens.
  • MS mass spectrometry
  • the resultant peptide hits from MS screening will be resynthesized by solid phase techniques. These peptides hits will be separately incubated with insulin, and this insulin complex will be subsequently tested for insulin receptor activation when compared to native insulin in a cell-based assay.
  • the envisioned assay will measure the level of phosphorylated Akt, a signaling intermediate downstream of the activated insulin receptor.
  • insulin activity will be monitored in the presence of the peptide hit and hyperglycemic levels of glucose. This will validate whether suppression of insulin activity by the hit peptides is glucose-responsive.
  • the formulation of delivery of insulin with peptide hits will be examined to determine whether a more efficient glucose-responsive trigger can be generated.
  • protein engineering we can explore tethering the peptide to insulin using a flexible linker to improve the binding and responsiveness of the compound.
  • multivalent peptide structures can be explored to evaluate whether the formation of higher order insulin-peptide aggregates could be used to improve the delivery and the potency of the therapy.
  • the insulin- peptide complexes will also be formulated with existing drug delivery platforms, for example polymeric nanoparticles, to optimize drug delivery kinetics and protect both the insulin and peptide from enzymatic degradation.
  • the insulin/peptide formulation will be injected subcutaneously or intravenously into streptazotocin-induced diabetic mice.
  • the blood glucose of these mice will be monitored to determine the efficacy and duration of glycemic correction, including probing the insulin-responsiveness by bolus glucose challenge of reversed mice, and monitoring for the appropriate dose to ensure long-term normoglycemia.
  • blood glucose will be monitored every 30 minutes after injection until 8 hours.
  • blood glucose will be monitored 4 times a day for 2 weeks. Promising candidates will be evaluated in additional models with help from Sanofi for both safety and efficacy.
  • Example 3 Synthesis and testing of insulin derivatives complexed with hydrophobic phenylboronic acid groups
  • Insulins with both long acting and glucose responsive properties were engineered in order to achieve improved glycemic control for diabetics.
  • Extended insulin release was achieved through conjugation of the protein to hydrophobic groups, which can ultimately be cleaved in response to glucose concentration.
  • the desired insulin analogs were synthesized via a three-step reaction sequence. First, native insulin was selectively modified with a clickable alkyne moiety at the B29 residue. A diol was then linked to the insulin via copper-free click chemistry. Finally, the diol group reacted with a
  • the modified insulin analogs were purified with a variety of physical methods and
  • Concanavalin-A Con-A
  • Con-A Concanavalin-A
  • diols/polysaccharides were harnessed to prepare dynamic, high molecular weight complexes [Kawamura et al, Colloids Surf B Biointerfaces 2011, Tang et al., Biotechnol. Bioeng. 2003, 82, 47-53]. These complexes disintegrate when glucose concentration increases, allowing insulin to be released.
  • Native insulin is not suitable for direct injection because of the 30 minute delay time for action (due to self-association) and overall short lasting effect [Berman, Diabetes Care 1980, 3, 266-269]. Accordingly, both long and short acting insulins have been developed to treat diabetic patients, whom need both types to maintain glycemic control [Esposito and Giugliano, Expert Opin. Biol. Ther. 2012, 12, 209-221].
  • the short acting form is injected just before a meal, and the long acting form is administrated twice per day to maintain a basal concentration of insulin.
  • Short-acting analogs are made via chemical modifications or by genetically modifying the insulin amino acid sequence (Lipsro, Aspart).
  • Bioconjugate Chem. 2005, 16, 615-620 These grafted compounds increase the in vivo circulation time of insulin due to serum albumin interactions, as well as increasing hydrodynamic volume. Additional studies where insulin was conjugated to albumin or polysaccharides, showed to also increase in vivo circulation time [Shechter et al, Bioconjugate Chem. 2005, 16, 913-920, Baudys et al, Bioconjugate Chem. 1998, 9, 176-183].
  • Glargine an insulin with an increased isoelectric point (IP 6.7)
  • IP 6.7 isoelectric point
  • Zinc-stapled insulin made via recombinant methods, has an increased number of zinc binding sites causing enhanced self-association properties [Phillips et al, J. Biol. Chem. 2010, 285, 11755-11759]. Both the glargine and zinc-stapled insulin analogs were shown to diffuse slowly from the subcutaneous injection site.
  • the objective of this work is to develop novel insulin formulations which have both long acting and glucose responsive properties without the use of a polymeric medium.
  • click chemistry was utilized and optimized for the chemical modification of native insulin.
  • First alkyne moieties were selectively conjugated onto the B29 lysine residue, which generated clickable insulin.
  • the alkyne group enabled efficient functionalization of the insulin with various azido-substituted diols.
  • a specifically designed azido substituted peptide featuring various dopamine groups was conjugated to the insulin.
  • the dopamine functional groups were then linked to hydrophobic phenylboronic acid derivatives via the formation of boronate ester.
  • the hydrophobic insulin formulations were studied to determine both the long acting properties and glucose responsiveness at physiological conditions through the reversible formation of the boronate ester.
  • the interaction between various diols and phenylboronic acids were studied with U B NMR, and the modified insulin analogs and their formulations were extensively investigated for their bioactivities, and capabilities of complexing with selected boronic acids in order to generate a realistic model. Further optimization of the system is needed to achieve further implementation of these formulations.
  • Propargyl dPEG NHS ester was purchased from Quanta Biodesign; DBCO NHS ester was purchased from Click Chemistry Tools. 8-
  • Azidoadenosine, 2,5-dioxopyrrolidin-l-yl tetradecanoate N-succinimidyl myristate was from Santa Cruz biotechnology.
  • Recombinant human insulin was purchased from Life Technologies Corporation.
  • 4-(N-cyclohexyl-N-(4- methoxybenzyl)sulfamoyl)phenylboronic acid (4NBA) was from Combi blocks.
  • 6-Azido-6-deoxy-d-galactose was purchased from CarboSynth Ltd. All organic solvents and other chemicals were purchased from Sigma Aldrich (US).
  • This system consisted of 2 1260 prep pumps, a 1260 prep ALS, a 1260 DAD DL UV light detector, and a 1260 FCPS fraction collector. Samples were run on a preparative C-18 column (Atlantis, Waters 250 mm X 25 mm) using acetonitrile (with 1.5 % acetic acid)/DDW; gradient was applied from 5:95 to 40:60; wavelength: 220 nm. Pure insulin samples were lyophilized and characterized via LC/MS Waters system; Acquity LC equipped with a QTof MS purchased together from Waters.
  • (+)-sodium L-ascorbate 300 umole was dissolved in 0.375 mL of DDW.
  • the various solutions were added to the insulin solution in the following order: (1) 1.2 mL of the TBTA solution was pre-complexed with 0.6 mL of the CuS0 4 solution; the complex was then added (2) The
  • the second approach to modify insulin with azido diols utilized dibenzocyclooctyne (DBCO) copper-free chemistry.
  • DBCO dibenzocyclooctyne
  • 1.5 mg/ml of the hormone was suspended in PBS along with 3 eq of various azido compounds; the reaction was carried out for 24 hour at 4 °C.
  • the crude product was purified via dialysis against DDW and samples were evaluated using LC-MS. 6G-DBCO-insulin, m/z [M+H] + 6383.0, [M+5H] 5+ 1277.43, [M+4H] 4+ 1596.57.
  • DP1-DBCO- insulin [M+H] + 6559.0, [M+5H] 5+ 1311.8, [M+4H] 4+ 1639.7.
  • DP3-DBCO- insulin [M+H] + 6144.0, [M+5H] 5+ 1428.8 and [M+4H] 4+ 1786.2.
  • DPI -peptide (azido-pentanoic-DOPA-GLY-NH 2 ) and DP3 peptide (azido-pentanoic-DOPA-GLY-DOPA-GLY-DOPA-GLY-NH 2 ) were synthesized using Fmoc protected amino acids Fmoc-3, 4-dihydroxy- phenylalanine, acetonitrile protected. Azido pentanoic acid was finally conjugated to the N-terminal in a procedure.
  • the correct molecular weight was determined by MALDI-TOF mass spectrometry (ABI model Voyager DESTR using sinapinic acid or alpha cyano-4-hydroxycinnamic acid as matrix) and purity was determined by analytical HPLC (Agilent model 1100).
  • Azido peptides were prepared at the Biopolymer Facility, located within the Koch Institute for Integrative Cancer Research at MIT.
  • aqueous/organic 2/1 v/v solution in order to enable co-solubility with hydrophobic phenylboronic acids.
  • Equal molar quantities of 4NBA and DP3- Insulin (0.5 mM) were dissolved in 2:1 acetonitrile: PBS and then the pH was modified to 7.5 or 8.5 by adding calculated amount of concentrated NaOH solution. The obtained complex was immediately frozen in liquid nitrogen and then freeze dried to generate a white solid.
  • selected diols (glucose, fructose, 4EC, 2HA, Azido-6-glactose, DPl-peptide and DP3-peptide) were formulated via the direct method.
  • DP3 -insulin (0.026 mM) was formulated with an aliphatic modified phenylboronic acid (C12-Nitro-PBA, 0.13 mM) in aqueous solution in the presence of 3 Zn 2+ per hexamer.
  • C12-Nitro-PBA aliphatic modified phenylboronic acid
  • DP3-Insulin was activated zinc in order to induce self-association; C12-Nitro-PBA was dissolved in similar solution and both solutions were mixed. Freshly prepared formulations were used for NMR analysis and for the in vivo study.
  • Samples tested for FTIR analysis were prepared using the direct complex method. However, instead of using a DP3 -insulin and a hydrophobic PBA samples were prepared with Fructose and 1TBA. 1TBA and fructose were selected to model this process due to their distinguish FTIR spectra. Samples were prepared at organic/aqueous solution with pH of 7.4 or 8.5. Controls made with no fructose were prepared under similar conditions (direct method). Bulk 1TB A, fructose and their solid mixtures were also evaluated. FTIR spectra were measured with Alpha FTIR spectrometer (Bruker optics Inc.). KBr plates of samples were measured in the transmission mode.
  • Insulin analogs were dissolved in phosphate buffer along with ZnCl 2 (3 per Hexamer), EDTA 0.1% w/v and glycerol 5% v/v. Samples were agitated in room temperature at 100 rpm for 30 min. Aliquots of the solutions were taken following centrifugation (10,000 rpm, 5 min). Measurements were carried out relative to a calibration curve that was obtained for each analog using an analytical HPLC as depicted above; however, for this procedure the mobile phase was acetonitrile:DDW containing 0.1% TFA.
  • Size distributions were measured using Zetasizer nano ZS dynamic light scattering (Malvern Instruments) equipped with a He-Ne laser at 633nm.
  • HeLa cells were maintained in growth media consisting of RPMI-1640 with L-glutamine, penicillin/streptomycin, and 10% heat inactivated fetal bovine serum (Invitrogen Corp.). For cell viability experiments, cells were seeded in 96-well plates at 10,000 cells per well and allowed to settle overnight.
  • the media was removed and replaced with 150 growth media and 50 ⁇ , of sample compound, dissolved in phosphate buffered saline and filtered through a 0.02- ⁇ Anotop filter (Whatman, Clifton, NJ 07014). After 72 hours growth media was removed and wells were washed once with Hanks Balanced Salt Solution, with calcium and magnesium (Invitrogen, Carlsbad, CA). MTS assay solution (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega Corp., Madison, WI), was added to each well according to provider's directions and allowed to incubate for 3 hours. The media was then transferred to a new plate before reading the absorbance at 490 nm.
  • MTS assay solution CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega Corp., Madison, WI
  • mice All procedures used in animal studies were approved by the Committee on Animal Care at MIT and were consistent with local, state, and federal regulations prior initiation of this research.
  • STZ-induced mice were purchased from the Jackson laboratory (MA, USA) (C57BL, male, 6 weeks).
  • Insulin formulations were freshly prepared by dissolving the appropriate insulin analog or the native form in PBS (0.1 M, pH 7.4, ZnCl 2 :3 Zn per hexamer) to a final concentration of 0.026mM (0.15 mg/ml for Native-insulin). The pH was adjusted to pH 7.4 in case C12-Nitro-PBA was added to the formulation.
  • Each tested group consisted of 4 STZ-induced diabetic mice; animals initial blood glucose levels were measured by bleeding their tails and collecting 2-5 ul of blood to a glucose test strip meter (Clarity Plus; Diagnosis Inc). Animals were then subcutaneous injected with 80 ul of the formulation (IX) or 40 ul (0.5X). Blood glucose levels were tested every 30 minutes over a span of 6-7 hrs. The animals were then humanly sacrificed at the end of the experiment. Data is presented is the average value of each time point for each 4 animals.
  • Human insulin has a single lysine residue within its primary structure; it was selected for modification at this specific ⁇ -amino group. There are also two other free amino groups located on the N-termini of both chains, but the amino group of B29 is more reactive than the other two in a basic anhydrous solution, and therefore can be selectively modified. Furthermore, conjugation at this location was previously found to preserve the hormone's in vivo bioactivity [Evans et al, Diabetes, Obes. Metab. 2011, 13, 677-684]. Native insulin was functionalized with an alkyne by reacting it with NHS activated propargyloxy propionate (Figure 15 (Scheme 1)). The conjugation reaction was very efficient and reached completion within 30 minutes at room temperature.
  • the modified insulin (Al- insulin) was purified with preparative HPLC. Insulin was also modified using DBCO-NHS esters and purified in a similar manner (DBCO-insulin). Both reactions were optimized for the molar equivalents of the modifying reagent. It was found that the desired modification requires a use of a single equivalent of alkyne; when two or more equivalents of NHS ester were added, the crude product mainly consisted of insulin functionalized with multiple conjugates. Purified Al-insulin and DBCO-insulin were characterized with LC-MS
  • Alkyne modified insulin was further modified with azido-terminated compounds.
  • the copper-catalyzed azide-alkyne cycloaddition is a well-known "click" chemistry tool and it is widely used in bio-conjugation [Ganesh et al, Chem.— Asian J. 2011, 6, 2670-2694].
  • Initial attempts to functionalize Al-insulin without the use of a chelate resulted with multiple modifications as reflected by the LC-MS data (supporting information). Therefore the process was amended [Hong et al, Angew. Chem., Int. Ed.
  • the chemical shift of boron in PBA changes when it is complexed with a diol.
  • the neutral trivalent PBA shows a chemical shift at about 10 ppm, while the boronate ester is shifted to -10 ppm. Therefore, the efficiency of complexation was estimated by monitoring the ratio of these two peaks.
  • the effect of pH on complexation and structure of diols was studied. As shown in Table 1, free glucose showed only minimal complexation at pH 7.4, with 11% at a 2: 1 ratio (diol/PBA). However, the complexation was significantly increased at elevated pH. Complexation was up to 50% by increasing 1 pH unit (pH 8.5). It also showed the azido groups at the anomeric position abolished the binding ability with PBA completely.
  • modified insulin with PBAs was investigated with a PBA conjugated resin. As shown in Figure 17, about 85% native insulin was recovered, while 6-galactose modified insulin had a recover less than 40%, DP1- insulin (peptide modified with a single dopamine group) had less than 10% recovery, and DP3 -insulin (three dopamine group) had less than 5%. Diol- modified insulin showed the strongest affinity to the resin, in agreement with the u B NMR data.
  • diols PBA concentration is 10 mM; diol concentration is with respect to used equivalence. Samples were dissolved in a mixture of acetonitrile and phosphate buffer saline 0.1 M and were tested at room temperature.
  • Formulations of modified insulin were further analyzed with UC
  • Native insulin was formulated with m-cresol and Zn 2+ . Native insulin mainly exists in the hexamers form (S.D 3.3) with minor populations of monomers and trimers (S.D 2.0). DBCO-insulin showed the presence of dimers, trimers, and in addition, large molecular weight aggregates (SD 4.5), demonstrating that analogs not only preserve their capacity to self- associate, but also have the ability form large structures as well.
  • DP3 -insulin was formulated with an aliphatic modified PBA (C12-nitro- PBA) in aqueous solution in the presence of Zn 2+ ( Figure 21 (Scheme 2)).
  • CI 2- nitro-PBA was synthesized because it has low ⁇ ⁇ of about 6.5. The low ⁇ ⁇ of the compound has increased solubility and optimal binding affinity with the
  • DP3-insulin under physiological conditions.
  • the complex formed is expected to induce intermolecular hydrophobic interactions between self-assonating insulins.
  • DP3 -insulin was formulated with Zn 2+ to induce self- association, and then mixed with C12-nitro-PBA in order to induce complexing via hydrophobic interaction.
  • Hydrophobic insulin formulations were freshly prepared by dissolving DP3 -insulin in phosphate buffer saline containing three Zn 2+ ions per hexamer. Ci2-nitro-PBA was dissolved in a similar solution and the complex was formed upon mixing at room temperature (supporting information). Controls that contain C12-insulin, native insulin and DP3 -insulin but no PBA were prepared similarly. Each group tested consisted of 4 STZ-induced diabetic mice that were subcutaneous injected with either 80 ⁇ (IX) or 40 ⁇ (0.5X) of the formulation. Blood glucose levels were monitored every 30 minutes over at least 6 hrs. The glucose level data that is presented here is an average of measurements taken from 4 different animals.
  • C12-insulin prepared by conjugating insulin to an aliphatic chain, demonstrated the expected [Hordern, Drugs Today 2006, 42, 505-517], long acting properties relative to native insulin.
  • DP3 -insulin (B) maintained low glucose level for least six hours, while the native insulin control shifts mice glucose levels back to a high glycemic index in less than 2 hours.
  • Complexing DP3-insulin with C12-Nitro-PBA and Zn 2+ was expected to generate hydrophobic interactions (Figure 21 (Scheme 2)), which would extend the formulation release time out of the injection site.
  • System glucose responsiveness can be optimized accordingly to address physiological glucose levels and the kinetics of insulin release.
  • reversible aggregates are generated by self-associating diol-insulins encapsulated by interacting aliphatic phenylboronic acids. Destabilizing these aggregates should occur according to the dynamic alteration within blood glucose levels typical in diabetic patients (2-6 mg/dL). It is postulated that the insulin diffusion rate out of a subcutaneous injection site is a balance between complexing forces to hydrophobic interactions.
  • PBA can complex with either glucose or insulin analogs with respect to their relative concentrations and with respect to the diol structures.
  • the hydrophobic interaction between insulin clusters is a function of the length of the aliphatic chain, the efficiency of the conjugation to insulin, and the nature of self-associating clusters (hexamers, dimers or other forms).
  • a study estimating the influence of tunable parameters such as the length of the aliphatic chain, the pKa of the boronic acid, the type of functionalized diols, and the number of diols per insulin can be used to engineer the system that meets the physiological window. Such studies should include developing a designated animal protocol that can help to characterize these parameters.
  • the resulting hydrophobic insulin formulation was a homogeneously clear and injectable solution, and was shown to have a long acting effect with respect to non-complexed analog.
  • This example describes a system for combinatorially generating oligomeric conjugates, attaching them to siR A, and evaluating
  • the system here functionalizes the monomers with thirteen nonpolar cyclic and acyclic hydrocarbon side chains, tertiary amines, cyclic amines with physiological pKa's, and both cyclic and acyclic neutral hydrophilic moieties.
  • the inclusion of a fluorescent monomer can create conjugates that can be used as imaging agents of delivery.
  • conjugates preferably will have molecular weights much larger than small molecules but slightly smaller than the siRNA molecules themselves.
  • trimeric conjugates that contain monomers with single-modified and dual-modified sidechains are preferred ( Figures 24A- 24C).
  • a variety of monomer backbones also referred to monomer building blocks) can be used to provide for more or fewer side chains and to account for reactivity differences between monomers. Some examples of alternatives are shown in Figure 25.
  • the oligomer conjugates can be evaluated and optimized for efficacious siRNA delivery. For example, conjugates that display greater than 50% protein knockdown in vitro (using mouse cancer cell lines) can be tested in mice for biodistribution and endogenous gene silencing in the liver and lung.
  • Immunogenicity can be determined by monitoring mouse cytokine levels.
  • Oligomers displaying combinations of delivery-based functionalities will exhibit increased siRNA delivery in vitro and in vivo. Oligomer conjugates of most interest will exhibit low immunogenicity and greater than 90% protein knockdown in vivo when administered at 1 mg/kg body weight.
  • the mechanism of oligomer-mediated siRNA delivery can be assessed in appropriate cells, such as primary mouse hepatocyte and primary mouse lung epithelial cells, by testing one or more of four different internalizations routes: clathrin-mediated endocytosis, caveolae-mediated endocytosis, charge-based cell penetration, and serum protein-mediated uptake.
  • the oligomer conjugates can be make use of more than one delivery route, which can aid in improved and efficient delivery.
  • the oligomer conjugates can also be evaluated for numerous properties, such as pKa and hydrophobicity. Correlation of these properties to the effectiveness of the oligomer conjugates can be used to guide optimization of the oligomer conjugates. For example, the properties and oligomer conjugate activity can be subjected to principal component analysis (PC A) to illuminate design principles [15, 16].
  • PC A principal component analysis
  • the oligomer conjugates represent new means of effectively delivering siRNA to cells and tissues as well as new tools for siRNA research and development of siRNA-based therapies.
  • siRNA small-interfering RNA
  • Exogenous siRNA sequences can utilize the cellular mechanism of RNA interference (RNAi) to catalyze the destruction of complementary protein- encoding R A sequences, resulting in sequence-specific gene silencing [3-6].
  • RNAi RNA interference
  • the prevalence of disease targets considered “undruggable” using small molecules or protein-based therapies underscores the importance of pursuing siRNA-based approaches to improve clinical outcomes for a wide range of diseases [1].
  • siRNA therapy A major obstacle to implementation of siRNA therapy is systemic delivery of the oligonucleotide in vivo [7].
  • Polymeric or liposomal approaches have progressed towards resolving this challenge but require excess delivery material relative to siRNA, leading to issues associated with toxicity and practicality [7-14].
  • An alternative approach involves one-to-one modification of siRNA with chemical or biological entities that can facilitate delivery. This approach overcomes the use of excess delivery material, making it an attractive strategy to facilitate cellular delivery while minimizing unfavorable biological responses [8-10].
  • Conjugation of biological motifs to siRNA has achieved mixed results with limitations to clinical implementation, while small chemical entities have to date proven inefficacious [8,11,12].
  • Cholesterol-conjugates siRNA is the most efficacious small molecule formulation, but this approach requires doses that are intractable for therapeutic use [11-13].
  • Cell-penetrating peptides are efficacious in assisting delivery of payloads, but are considerably immunogenic due to the use of non-human peptides sequences [8].
  • a fully synthetic, peptide-mimetic conjugate system can capture the delivery potential of large polymeric molecules while displaying low immunogenicity at therapeutically relevant doses.
  • Nanoparticle formulations are an efficacious siRNA delivery agents, but there has been no investigation into whether the chemical functionalities that facilitate efficient delivery can be translated into smaller distinct chemical entities that can serve as covalently attached conjugates.
  • the system described here uses these delivery-biasing chemical moieties in a synthetic oligomeric approach to develop siRNA delivery conjugates.
  • a defined set of delivery-biased building blocks were devised to serve as monomers, with the monomers used to build trimeric oligomers.
  • the combinatorial pairing of delivery-relevant functionalities can generate thousands of of uniqu oligomers with promising delivery potential.
  • This approach makes generation of efficient delivery of siRNA easier and allows analysis of the structure-function relationships of the oligomers to elucidate the most salient molecular properties for efficacious delivery.
  • This approach is the first time multiple delivery-relevant functionalities have been brought together in an oligomeric framework to identify optimal delivery agents while illuminating properties that govern delivery. Beyond their use for treatment of patients, the development of synthetic delivery conjugates are also useful tools for siRNA research and to provide understanding of chemical properties required to overcome cellular barriers.
  • Type 1 diabetic patients must adhere to a daily regimen of blood glucose monitoring and insulin injections to manage the disease. This management is often complicated by insufficient patient compliance [46] which leads to deregulation of their plasma glucose that can result in heart disease,
  • An injectable, self-regulating insulin release formulation is a promising approach to mitigate the complications resulting from poor patient compliance and improve overall life quality.
  • Short-acting insulin is utilized prior to a meal, with long-acting insulin administered twice per day to maintain appropriate basal insulin levels in the body.
  • Short-acting analogs are products of either covalent modification or by genetically modifying the insulin amino acid sequence (Lipsro, Aspart). These modifications reduce the hexamerization state of the insulin, increasing its bioavailability [50, 51].
  • long-acting formulations promote aggregated states and have been achieved by conjugation of native insulin with protamine (NPH) or a large molar excess of zinc (Lente) [52].
  • Other long-acting analogs have been prepared by covalent modification with polyethylene glycol (PEG) [53, 54], fatty acids [55], (detimer) or bile acids [56]. These analogs achieve higher in vivo circulation times due to serum albumin interactions. Additionally, direct conjugation of insulin to albumin or polysaccharide chains have shown an increased circulation times [57, 58].
  • Astriab-Fisher, A.; Sergueev, D. S.; Fisher, M.; Shaw, B. R; Juliano, R. 1. Antisense inhibition of P-glycoprotein expression using peptide- oligonucleotide conjugates. Biochem Pharmacol 2000, 60 (I), 83-90. Chiu, Y. L.; Ali, A.; Chu, C. Y.; Cao, H.; Rana, T. M., Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells. Chem Biol 2004, 11 (8), 1165-75.
  • siRNA of brain cancer with receptor targeting and avidin-biotin technology Pharm Res 2007, 24 (12), 2309-16.

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Abstract

L'invention concerne des composés, des compositions et des méthodes de libération "intelligente" d'un agent thérapeutique, prophylactique ou diagnostique, telle que la libération d'insuline médiée par le glucose au moyen de dérivés d'insuline sensibles au glucose. Les dérivés d'insuline fixent le sérum-albumine ou s'agglomèrent in vivo. Les dérivés d'insuline se dissocient efficacement pour libérer l'insuline dans un état hyperglycémique, la complexation du glucose en élément sensible au glucose modifiant les propriétés du dérivé d'insuline menant à la dissociation. Les composés, compositions, et méthodes permettent la mise en oeuvre d'une stratégie de libération pour la gestion du diabète à la fois auto-régulée et à long terme.
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WO2016149222A2 (fr) 2015-03-13 2016-09-22 Case Western Reserve University Analogues de l'insuline contenant un commutateur de conformation régulé par le glucose
WO2016149222A3 (fr) * 2015-03-13 2016-11-03 Case Western Reserve University Analogues de l'insuline contenant un commutateur de conformation régulé par le glucose
US20180057559A1 (en) * 2015-03-13 2018-03-01 Case Western Reserve University Insulin analogues with a glucose-regulated conformational switch
JP2018513843A (ja) * 2015-03-13 2018-05-31 ケース ウェスタン リザーブ ユニバーシティＣａｓｅ Ｗｅｓｔｅｒｎ Ｒｅｓｅｒｖｅ Ｕｎｉｖｅｒｓｉｔｙ グルコース制御立体配座スイッチを含むインスリン類似体
US10584156B2 (en) 2015-03-13 2020-03-10 Case Western Reserve University Insulin analogues with a glucose-regulated conformational switch
AU2016233430B2 (en) * 2015-03-13 2020-06-04 Case Western Reserve University Insulin analogues containing a glucose-regulated conformational switch
KR102677852B1 (ko) 2015-03-13 2024-06-25 케이스 웨스턴 리저브 유니버시티 글루코스 조절된 구조 전환을 포함하는 인슐린 유사체
JP2021178833A (ja) * 2015-03-13 2021-11-18 ケース ウェスタン リザーブ ユニバーシティCase Western Reserve University グルコース制御立体配座スイッチを含むインスリン類似体
WO2017070617A1 (fr) * 2015-10-21 2017-04-27 Case Western Reserve University Analogues de l'insuline modifiés avec un diol contenant un commutateur de conformation régulé par le glucose
US11186595B2 (en) 2017-11-09 2021-11-30 Novo Nordisk A/S Glucose-sensitive albumin-binding derivatives
US11767332B2 (en) 2017-11-09 2023-09-26 Novo Nordisk A/S Glucose-sensitive albumin-binding derivatives
TWI717245B (zh) * 2019-03-29 2021-01-21 丹麥商諾佛 儂迪克股份有限公司 葡萄糖敏感型胰島素衍生物
WO2020201041A3 (fr) * 2019-03-29 2020-11-19 Novo Nordisk A/S Dérivés d'insuline sensibles au glucose
KR20210148143A (ko) * 2019-03-29 2021-12-07 노보 노르디스크 에이/에스 포도당 민감성 인슐린 유도체
WO2020201041A2 (fr) 2019-03-29 2020-10-08 Novo Nordisk A/S Dérivés d'insuline sensibles au glucose
KR102507156B1 (ko) 2019-03-29 2023-03-09 노보 노르디스크 에이/에스 포도당 민감성 인슐린 유도체
EP4003426A4 (fr) * 2019-07-31 2023-07-05 Thermalin Inc. Analogues de l'insuline à commutateur de conformation régulé par le glucose
WO2021202802A1 (fr) * 2020-03-31 2021-10-07 Protomer Technologies Inc. Conjugués pour une réactivité sélective à des diols vicinaux
GB2610490A (en) * 2020-03-31 2023-03-08 Protomer Tech Inc Conjugates for selective responsiveness to vicinal diols
WO2022109078A1 (fr) * 2020-11-19 2022-05-27 Protomer Technologies Inc. Composés aromatiques contenant du bore et analogues d'insuline

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